Treatment and diagnosis of prostate cancer

ABSTRACT

The present invention is directed to the use of antibodies or binding portions thereof, probes, ligands, or other biological agents which either recognize an extracellular domain of prostate specific membrane antigen or bind to and are internalized with prostate specific membrane antigen. These biological agents can be labeled and used for detection of normal, benign hyperplastic, and cancerous prostate epithelial cells or portions thereof. They also can be used alone or bound to a substance effective to ablate or kill such cells as a therapy for prostate cancer. Also disclosed are four hybridoma cell lines, each of which produces a monoclonal antibody recognizing extracellular domains of prostate specific membrane antigens of normal, benign hyperplastic, and cancerous prostate epithelial cells or portions thereof.

The present application is a divisional of application Ser. No.08/838,682, filed on Apr. 9, 1997, issued as U.S. Pat. No. 6,107,090,which claims the benefit of U.S. Provisional Patent Application Ser. No.60/016,976, filed May 6, 1996, and U.S. Provisional Patent ApplicationSer. No. 60/022,125, filed Jul. 18, 1996.

FIELD OF THE INVENTION

The present invention relates to the treatment and diagnosis of prostatecancer with biological agents.

BACKGROUND OF THE INVENTION

Prostate cancer is the most common cancer in men with an estimated317,000 cases in 1996 in the United States. It is the second leadingcause of death among men who die from neoplasia with an estimated 40,000deaths per year. Prompt detection and treatment is needed to limitmortality caused by prostate cancer.

Detection of Prostate Cancer

When it metastasizes, prostatic cancer has a distinct predilection forbone and lymph nodes. Saitoh et al., “Metastatic Patterns of ProstaticCancer. Correlation Between Sites And Number Of Organs Involved,”Cancer, 54:3078-3084 (1984). At the time of clinical diagnosis, as manyas 25% of patients have bone metastasis demonstrable by radionuclidescans. Murphy, G. P., et al., “The National Survey Of Prostate Cancer InThe United States By The American College Of Surgeons,” J. Urol.,127:928-939 (1982). Accurate clinical evaluation of nodal involvementhas proven to be difficult. Imaging techniques such as computedtomography (“CT”) or magnetic resonance (“MR”) imaging are unable todistinguish metastatic prostate cancer involvement of lymph nodes bycriterion other than size (i.e., >1 cm). Therefore, by definition, theseimaging modalities are inherently insensitive in the detection of smallvolume (<1 cm) disease as well as non-specific in the detection oflarger volume adenopathy. A recent study assessed the accuracy of MR inpatients with clinically localized prostate cancer. Rifkin et al.,“Comparison Of Magnetic Resonance Imaging And Ultrasonography In StagingEarly Prostate Cancer,” N. Engel. J. Med., 323:621-626 (1990). In thisstudy, 194 patients underwent an MR and 185 of these patients had alymph node dissection. 23 (13%) patients had pathologically involvedlymph nodes. MR was suspicious in only 1 of these 23 cases resulting ina sensitivity of 4%. Similar results have also been noted with CT scans.Gasser et al., “MRI And Ultrasonography In Staging Prostate Cancer,” N.Engl. J. Med. (Correspondence) 324(7):49-495 (1991).

The elevation of serum acid phosphatase activity in patients havingmetastasized prostate carcinoma was first reported by Gutman et al., J.Clin. Invest 17:473 (1938). In cancer of the prostate, prostatic acidphosphatase is released from the cancer tissue into the blood streamwith the result that the total serum acid phosphatase level can begreatly increased above normal values. Numerous studies of this enzymeand its relation to prostatic cancer have been made since that time,e.g. Yam, Amer. J. Med. 56:604 (1974). However, the measurement of serumacid phosphatase is elevated in about 65-90 percent of patients havingcarcinoma of the prostate with bone metastasis; in about 30 percent ofpatients without roentgenological evidence of bone metastasis; and inabout only 5-10 percent of patients lacking clinically demonstrablemetastasis.

Prior art attempts to develop a specific test for prostatic acidphosphatase have met with only limited success, because techniques whichrely on enzyme activity on a so-called “specific” substrate cannot takeinto account other biochemical and immunochemical differences among themany acid phosphatases which are unrelated to enzyme activity ofprostate origin. In the case of isoenzymes, i.e. genetically definedenzymes having the same characteristic enzyme activity and a similarmolecular structure but differing in amino acid sequences and/or contentand, therefore, immunochemically distinguishable, it would appearinherently impossible to distinguish different isoenzyme forms merely bythe choice of a particular substrate. It is, therefore, not surprisingthat none of these prior art methods is highly specific for the directdetermination of prostatic acid phosphatase activity; e.g. see Cancer5:236 (1952); J. Lab. Clin. Med. 82:486 (1973); Clin. Chem. Acta. 44:21(1973); and J. Physiol. Chem. 356:1775 (1975).

In addition to the aforementioned problems of non-specificity whichappear to be inherent in many of the prior art reagents employed for thedetection of prostate acid phosphatase, there have been reports ofelevated serum acid phosphatase associated with other diseases, whichfurther complicates the problem of obtaining an accurate clinicaldiagnosis of prostatic cancer. For example, Tuchman et al., Am. J. Med.27:959 (1959) noted that serum acid phosphatase levels appear to beelevated in patients with Gaucher's disease.

Due to the inherent difficulties in developing a “specific” substratefor prostate acid phosphatase, several researchers have developedimmunochemical methods for the detection of prostate acid phosphatase.However, the previously reported immunochemical methods have drawbacksof their own which have precluded their widespread acceptance. Forexample, Shulman et al., Immunology 93:474 (1964) described animmuno-diffusion test for the detection of human prostate acidphosphatase. Using antisera prepared from a prostatic fluid antigenobtained by rectal massage from patients with prostatic disease, nocross-reactivity precipitin line was observed in the double diffusiontechnique against extracts of normal kidney, testicle, liver, and lung.However, this method has the disadvantages of limited sensitivity, evenwith the large amounts of antigen employed, and of employing antiserawhich may cross-react with other, antigenically unrelated serum proteincomponents present in prostatic fluid.

WO 79/00475 to Chu et. al. describes a method for the detection ofprostatic acid phosphatase isoenzyme patterns associated with prostaticcancer which obviates many of the above drawbacks. However, practicalproblems are posed by the need for a source of cancerous prostatictissue from which the diagnostically relevant prostatic acid phosphataseisoenzyme patterns associated with prostatic cancer are extracted forthe preparation of antibodies thereto.

In recent years, considerable effort has been spent to identify enzymeor antigen markers for various types of malignancies with the viewtowards developing specific diagnostic reagents. The ideal tumor markerwould exhibit, among other characteristics, tissue or cell-typespecificity. Previous investigators have demonstrated the occurrence ofhuman prostate tissue-specific antigens.

Treatment of Prostate Cancer

As described in W. J. Catalona, “Management of Cancer of the Prostate,”New Engl. J. Med., 331(15):996-1004 (1994), the management of prostatecancer can be achieved by watchful waiting, curative treatment, andpalliation.

For men with a life expectancy of less than 10 years, watchful waitingis appropriate where low-grade, low-stage prostate cancer is discoveredat the time of a partial prostatectomy for benign hyperplasia. Suchcancers rarely progress during the first five years after detection. Onthe other hand, for younger men, curative treatment is often moreappropriate.

Where prostate cancer is localized and the patient's life expectancy is10 years or more, radical prostatectomy offers the best chance foreradication of the disease. Historically, the drawback of this procedureis that most cancers had spread beyond the bounds of the operation bythe time they were detected. However, the use of prostate-specificantigen testing has permitted early detection of prostate cancer. As aresult, surgery is less extensive with fewer complications. Patientswith bulky, high-grade tumors are less likely to be successfully treatedby radical prostatectomy.

After surgery, if there are detectable serum prostate-specific antigenconcentrations, persistent cancer is indicated. In many cases,prostate-specific antigen concentrations can be reduced by radiationtreatment. However, this concentration often increases again within twoyears.

Radiation therapy has also been widely used as an alternative to radicalprostatectomy. Patients generally treated by radiation therapy are thosewho are older and less healthy and those with higher-grade, moreclinically advanced tumors. Particularly preferred procedures areexternal-beam therapy which involves three dimensional, conformalradiation therapy where the field of radiation is designed to conform tothe volume of tissue treated; interstitial-radiation therapy where seedsof radioactive compounds are implanted using ultrasound guidance; and acombination of external-beam therapy and interstitial-radiation therapy.

For treatment of patients with locally advanced disease, hormonaltherapy before or following radical prostatectomy or radiation therapyhas been utilized. Hormonal therapy is the main form of treating menwith disseminated prostate cancer. Orchiectomy reduces serumtestosterone concentrations, while estrogen treatment is similarlybeneficial. Diethylstilbestrol from estrogen is another useful hormonaltherapy which has a disadvantage of causing cardiovascular toxicity.When gonadotropin-releasing hormone agonists are administeredtestosterone concentrations are ultimately reduced. Flutamide and othernonsteroidal, anti-androgen agents block binding of testosterone to itsintracellular receptors. As a result, it blocks the effect oftestosterone, increasing serum testosterone concentrations and allowspatients to remain potent—a significant problem after radicalprostatectomy and radiation treatments.

Cytotoxic chemotherapy is largely ineffective in treating prostatecancer. Its toxicity makes such therapy unsuitable for elderly patients.In addition, prostate cancer is relatively resistant to cytotoxicagents.

Use of Monoclonal Antibodies in Prostate Cancer Detection and Treatment

Theoretically, radiolabeled monoclonal antibodies (“mAbs”) offer thepotential to enhance both the sensitivity and specificity of detectingprostatic cancer within lymph nodes and elsewhere. While many mAbs havepreviously been prepared against prostate related antigens, none ofthese mAbs were specifically generated with an imaging objective inmind. Nevertheless, the clinical need has led to evaluation of some ofthese mAbs as possible imaging agents. Vihko et al., “Radioimaging ofProstatic Carcinoma With Prostatic Acid Phosphatase—SpecificAntibodies,” Biotechnology in Diagnostics, 131-134 (1985); Babaian etal., “Radioimmunological Imaging of Metastatic Prostatic Cancer With111-Indium-Labeled Monoclonal Antibody PAY 276,” J. Urol., 137:439-443(1987); Leroy et al., “Radioimmunodetection Of Lymph Node Invasion InProstatic Cancer. The Use Of Iodine 123 (123-I)-Labeled MonoclonalAnti-Prostatic Acid Phosphatase (PAP) 227 A F (ab′) 2 Antibody FragmentsIn Vivo,” Cancer, 64:1-5 (1989); Meyers et al., “Development OfMonoclonal Antibody Imaging Of Metastatic Prostatic Carcinoma,” TheProstate, 14:209-220 (1989).

In some cases, the monoclonal antibodies developed for detection and/ortreatment of prostate cancer recognize antigens specific to malignantprostatic tissues. Such antibodies are thus used to distinguishmalignant prostatic tissue (for treatment or detection) from benignprostatic tissue. See U.S. Pat. No. 4,970,299 to Bazinet et al. and U.S.Pat. No. 4,902,615 to Freeman et al.

Other monoclonal antibodies react with surface antigens on all prostateepithelial cells whether cancerous or benign. See U.S. Pat. Nos.4,446,122 and Re 33,405 to Chu et al., U.S. Pat. No. 4,863,851 to McEwanet al., and U.S. Pat. No. 5,055,404 to Ueda et al. However, the antigensdetected by these monoclonal antibodies are present in the blood and,therefore, compete with antigens at tumor sites for the monoclonalantibodies. This causes background noise which makes the use of suchantibodies inadequate for in vivo imaging. In therapy, such antibodies,if bound to a cytotoxic agent, could be harmful to other organs.

Horoszewicz et al., “Monoclonal Antibodies to a New Antigenic Marker inEpithelial Prostatic Cells and Serum of Prostatic Cancer Patients,”Anticancer Research, 7:927-936 (1987) (“Horoszewicz”) and U.S. Pat. No.5,162,504 to Horoszewicz describe an antibody, designated 7E11, whichrecognizes prostate specific membrane antigen (“PSMA”). Israeli et al.,“Molecular Cloning of a Complementary DNA Encoding a Prostate-specificMembrane Antigen,” Cancer Research, 53:227-230 (1993) (“Israeli”)describes the cloning and sequencing of PSMA and reports that PSMA isprostate-specific and shows increased expression levels in metastaticsites and in hormone-refractory states. Other studies have indicatedthat PSMA is more strongly expressed in prostate cancer cells relativeto cells from the normal prostate or from a prostate with benignhyperplasia. Furthermore, PSMA is not found in serum (Troyer et al.,“Detection and Characterization of the Prostate-Specific MembraneAntigen (PSMA) in Tissue Extracts and Body Fluids,” Int. J. Cancer,62:552-558 (1995)).

These characteristics make PSMA an attractive target for antibodymediated targeting for imaging and therapy of prostate cancer. Imagingstudies using indium-labeled 7E11 have indicated that the antibodylocalizes quite well to both the prostate and to sites of metastasis. Inaddition, 7E11 appears to have clearly improved sensitivity fordetecting lesions compared to other currently available imagingtechniques, such as CT and MR imaging or bone scan. Bander, “CurrentStatus of Monoclonal Antibodies for Imaging and Therapy of ProstateCancer,” Sem. In Oncology, 21:607-612 (1994).

However, the use of 7E11 and other known antibodies to PSMA to mediateimaging and therapy has several disadvantages. First, PSMA is anintegral membrane protein known to have a short intracellular tail and along extracellular domain. Biochemical characterization and mapping(Troyer et al., “Biochemical Characterization and Mapping of the7E11-C5.3 Epitope of the Prostate-specific Membrane Antigen,” Urol.Oncol., 1:29-37 (1995)) have shown that the epitope or antigenic site towhich the 7E11 antibody binds is present on the intracellular portion ofthe molecule. Because antibody molecules do not, under normalcircumstances, cross the cell membrane unless they bind to theextracellular portion of a molecule and become translocatedintracellularly, the 7E11 antibody does not have access to its antigenictarget site in an otherwise healthy, viable cell.

Consequently, imaging using 7E11 is limited to the detection of deadcells within tumor deposits. Additionally, the therapeutic use of the7E11 antibody is limited, because only cells that are already dead ortissue containing a large proportion of dead cells can be effectivelytargeted.

The present invention is directed to overcoming the deficiencies ofprior art antibodies in diagnosing and treating prostate cancer.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to a method of ablating orkilling normal, benign hyperplastic, and cancerous prostate epithelialcells. The process involves providing a biological agent whichrecognizes an extracellular domain of prostate specific membraneantigen. The biological agent can be used alone or can be bound to asubstance effective to kill the cells upon binding of the biologicalagent to the cells. These biological agents are then contacted with thecells under conditions effective to permit both binding of thebiological agent to the extracellular domain of the prostate specificmembrane antigen and killing or ablating of the cells.

In another particularly preferred embodiment of the method of ablatingor killing normal, benign hyperplastic, and cancerous prostateepithelial cells in accordance with the present invention, thebiological agent binds to and is internalized with the prostate specificmembrane antigen of such cells. Preferred biological agents for use inthe method of ablating or killing normal, benign hyperplastic, andcancerous prostate epithelial cells in accordance with the presentinvention are antibodies or binding portions thereof, probes, orligands.

Another aspect of the present invention relates to a method of detectingnormal, benign hyperplastic, and cancerous prostate epithelial cells orportions thereof in a biological sample. This method involves providinga biological agent which binds to an extracellular domain of prostatespecific membrane antigen. The biological agent is bound to a labeleffective to permit detection of the cells or portions thereof uponbinding of the biological agent to the cells or portions thereof. Thebiological sample is contacted with the biological agent having a labelunder conditions effective to permit binding of the biological agent tothe extracellular domain of the prostate specific membrane antigen ofany of the cells or portions thereof in the biological sample. Thepresence of any cells or portions thereof in the biological sample isdetected by detection of the label.

In a particularly preferred embodiment of the method of detectingnormal, benign hyperplastic, and cancerous prostate epithelial cells inaccordance with the present invention, the biological agent binds to andis internalized with the prostate specific membrane antigen of suchcells. Preferred biological agents for use in the method of detectingnormal, benign hyperplastic, and cancerous prostate epithelial cells inaccordance with the present invention are antibodies or binding portionsthereof, probes, or ligands.

Another aspect of the present invention pertains to a biological agentthat recognizes an extracellular domain of prostate specific membraneantigen. In a preferred embodiment, the isolated biological agent bindsto and is internalized with the prostate specific membrane antigen.Preferred isolated biological agents which recognize an extracellulardomain of prostate specific membrane antigen in accordance with thepresent invention are isolated antibodies or binding portions thereof,probes, or ligands. Hybridoma cell lines that produce monoclonalantibodies of these types are also disclosed.

The biological agents of the present invention recognize theextracellular domain of antigens of normal, benign hyperplastic, andcancerous prostate epithelial cells. Unlike the 7E11 antibody, whichrecognizes an epitope of prostate-associated antigens which are exposedextracellularly only after cell lysis, the biological agents of thepresent invention bind to antigenic epitopes which are extracellularlyexposed in living prostate cells. Using the biological agents of thepresent invention, living, unfixed normal, benign hyperplastic, andcancerous prostate epithelial cells can be targeted, which makestreatment and diagnosis more effective. In a preferred embodiment, thebiological agents of the present invention also bind to and areinternalized with the prostate specific membrane antigen, which permitsthe therapeutic use of intracellularly acting cytotoxic agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an immuno-electron micrograph of gold-labeled monoclonalantibody J591 on the surface of LNCaP cells prior to incubation.

FIG. 2 is an immuno-electron micrograph of gold-labeled monoclonalantibody J591 after 5 minutes incubation at 37° C. LNCaP cells.

FIG. 3 is an immuno-electron micrograph of gold-labeled monoclonalantibody J591 after 10 minutes incubation at 37° C. LNCaP cells.

FIG. 4 is an immuno-electron micrograph of gold-labeled monoclonalantibody J591 after 15 minutes incubation at 37° C. LNCaP cells.

FIG. 5 is an immuno-electron micrograph of gold-labeled monoclonalantibody J591 after 15 minutes at 37° C. showing J591 within endosomes.

FIG. 6 summarizes the sequencing strategy of the heavy chain ofmonoclonal antibody J591.

FIG. 7 shows the nucleotide sequence of the heavy chain of monoclonalantibody J591 (designated SEQ. ID. No. 1), the nucleotide sequence ofthe corresponding reverse, non-coding strand (designated SEQ. ID. No.2), and the corresponding deduced amino acid sequences (designated SEQ.ID. Nos. 3, 4, and 5).

FIG. 8 is a comparison of the heavy chain of monoclonal antibody J591with the consensus sequence for Mouse Heavy Chains Subgroup IIA(designated SEQ ID No. 20).

FIG. 9 summarizes the sequencing strategy of the kappa light chain ofmonoclonal antibody J591.

FIG. 10 shows the nucleotide sequences of the kappa light chain ofmonoclonal antibody J591 (designated SEQ. ID. No. 9), the nucleotidesequence of the corresponding reverse, non-coding strand (designatedSEQ. ID. No. 10), and the corresponding deduced amino acid sequence(designated SEQ. ID. Nos. 11, 12, and 13).

FIG. 11 is a comparison of the kappa light chain of monoclonal antibodyJ591 with the consensus sequence for Mouse Kappa Chains Subgroup V(designated SEQ ID No. 21).

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention relates to a method of ablating orkilling normal, benign hyperplastic, and cancerous prostate epithelialcells. The process involves providing a biological agent, such as anantibody or binding portion thereof, probe, or ligand, which binds to anextracellular domain of prostate specific membrane antigen of (i.e., aportion of prostate specific membrane antigen which is external to) suchcells. The biological agent can be used alone or can be bound to asubstance effective to kill the cells upon binding of the biologicalagent to the cells. These biological agents are then contacted with thecells under conditions effective to permit both binding of thebiological agent to the extracellular domain of the prostate specificmembrane antigen and killing or ablating of the cells. In its preferredform, such contacting is carried out in a living mammal by administeringthe biological agent to the mammal under conditions effective to permitboth binding of the biological agent to the extracellular domain of theprostate specific membrane antigen and killing or ablating of the cells.Such administration can be carried out orally or parenterally.

In a particularly preferred embodiment of the method of ablating orkilling normal, benign hyperplastic, and cancerous prostate epithelialcells in accordance with the present invention, the biological agentbinds to and is internalized with the prostate specific membrane antigenof such cells. Again, the biological agent can be used alone.Alternatively, the biological agent can be bound to a substanceeffective to kill the cells upon binding of the biological agent toprostate specific membrane antigen and upon internalization of thebiological agent with the prostate specific membrane antigen.

The mechanism by which the biological agent is internalized with theprostate specific membrane antigen is not critical to the practice ofthe present invention. For example, the biological agent can induceinternalization of the prostate specific membrane antigen.Alternatively, internalization of the biological agent can be the resultof routine internalization of prostate specific membrane antigen.

Another aspect of the present invention relates to a method of detectingnormal, benign hyperplastic, and cancerous epithelial cells or portionsthereof in a biological sample. This method involves providing abiological agent, such as an antibody or binding portion thereof, probe,or ligand, which binds to an extracellular domain of prostate specificmembrane antigen of such cells. The biological agent is bound to a labeleffective to permit detection of the cells or portions (e.g., prostatespecific membrane antigen or fragments thereof liberated from suchnormal, benign hyperplastic, and cancerous cells) thereof upon bindingof the biological agent to the cells or portions thereof. The biologicalsample is contacted with the biological agent having a label underconditions effective to permit binding of the biological agent to theextracellular domain of the prostate specific membrane antigen of any ofthe cells or portions thereof in the biological sample. The presence ofany cells or portions thereof in the biological sample is detected bydetection of the label. In its preferred form, such contacting iscarried out in a living mammal and involves administering the biologicalagent to the mammal under conditions effective to permit binding of thebiological agent to the prostate specific membrane antigen of any of thecells or portions thereof in the biological sample. Again, suchadministration can be carried out orally or parenterally.

The method of the present invention can be used to screen patients fordiseases associated with the presence of normal, benign hyperplastic,and cancerous epithelial cells or portions thereof. Alternatively, itcan be used to identify the recurrence of such diseases, particularlywhen the disease is localized in a particular biological material of thepatient. For example, recurrence of prostatic disease in the prostaticfossa may be encountered following radical prostatectomy. Using themethod of the present invention, this recurrence can be detected byadministering a short range radiolabeled antibody to the mammal and thendetecting the label rectally, such as with a transrectal detector probe.

Alternatively, the contacting step can be carried out in a sample ofserum or urine or other body fluids, such as to detect the presence ofPSMA in the body fluid. When the contacting is carried out in a serum orurine sample, it is preferred that the biological agent recognizesubstantially no antigens circulating in the blood other than PSMA.Since intact prostate cells do not excrete or secrete PSMA into theextracellular environment, detecting PSMA in serum, urine, or other bodyfluids generally indicates that prostate cells are being lysed. Thus,the biological agents and methods of the present invention can be usedto determine the effectiveness of a prostate cancer treatment protocolby monitoring the level of PSMA in serum, urine or other body fluids.

In a particularly preferred embodiment of the method of detectingnormal, benign hyperplastic, and cancerous prostate epithelial cells inaccordance with the present invention, the biological agent, such as theantibody or binding portion thereof, probe, or ligand, binds to and isinternalized with the prostate specific membrane antigen of such cells.Again, the biological agent is bound to a label effective to permitdetection of the cells or portions thereof upon binding of thebiological agent to and internalization of the biological agent with theprostate specific membrane antigen.

As indicated above, biological agents suitable for either killing,ablating, or detecting normal, benign hyperplastic, and cancerousprostate epithelial cells include antibodies, such as monoclonal orpolyclonal antibodies. In addition, antibody fragments, half-antibodies,hybrid derivatives, probes, and other molecular constructs may beutilized. These biological agents, such as antibodies, binding portionsthereof, probes, or ligands, bind to extracellular domains of prostatespecific membrane antigens or portions thereof in normal, benignhyperplastic, and cancerous prostate epithelial cells. As a result, thebiological agents bind to all such cells, not only to cells which arefixed or cells whose intracellular antigenic domains are otherwiseexposed to the extracellular environment. Consequently, binding of thebiological agents is concentrated in areas where there are prostateepithelial cells, irrespective of whether these cells are fixed orunfixed, viable or necrotic. Additionally or alternatively, thesebiological agents, such as antibodies, binding portions thereof, probes,or ligands, bind to and are internalized with prostate specific membraneantigens or portions thereof in normal, benign hyperplastic, andcancerous prostate epithelial cells.

Monoclonal antibody production may be effected by techniques which arewell-known in the art. Basically, the process involves first obtainingimmune cells (lymphocytes) from the spleen of a mammal (e.g., mouse)which has been previously immunized with the antigen of interest eitherin vivo or in vitro. The antibody-secreting lymphocytes are then fusedwith (mouse) myeloma cells or transformed cells, which are capable ofreplicating indefinitely in cell culture, thereby producing an immortal,immunoglobulin-secreting cell line. The resulting fused cells, orhybridomas, are cultured, and the resulting colonies screened for theproduction of the desired monoclonal antibodies. Colonies producing suchantibodies are cloned, and grown either in vivo or in vitro to producelarge quantities of antibody. A description of the theoretical basis andpractical methodology of fusing such cells is set forth in Kohler andMilstein, Nature 256:495 (1975), which is hereby incorporated byreference.

Mammalian lymphocytes are immunized by in vivo immunization of theanimal (e.g., a mouse) with the protein or polypeptide of the presentinvention. Such immunizations are repeated as necessary at intervals ofup to several weeks to obtain a sufficient titer of antibodies.Following the last antigen boost, the animals are sacrificed and spleencells removed.

Fusion with mammalian myeloma cells or other fusion partners capable ofreplicating indefinitely in cell culture is effected by standard andwell-known techniques, for example, by using polyethylene glycol (“PEG”)or other fusing agents (See Milstein and Kohler, Eur. J. Immunol. 6:511(1976), which is hereby incorporated by reference). This immortal cellline, which is preferably murine, but may also be derived from cells ofother mammalian species, including but not limited to rats and humans,is selected to be deficient in enzymes necessary for the utilization ofcertain nutrients, to be capable of rapid growth, and to have goodfusion capability. Many such cell lines are known to those skilled inthe art, and others are regularly described.

Procedures for raising polyclonal antibodies are also well known.Typically, such antibodies can be raised by administering the protein orpolypeptide of the present invention subcutaneously to New Zealand whiterabbits which have first been bled to obtain pre-immune serum. Theantigens can be injected at a total volume of 100 μl per site at sixdifferent sites. Each injected material will contain syntheticsurfactant adjuvant pluronic polyols, or pulverized acrylamide gelcontaining the protein or polypeptide after SDS-polyacrylamide gelelectrophoresis. The rabbits are then bled two weeks after the firstinjection and periodically boosted with the same antigen three timesevery six weeks. A sample of serum is then collected 10 days after eachboost. Polyclonal antibodies are then recovered from the serum byaffinity chromatography using the corresponding antigen to capture theantibody. Ultimately, the rabbits are euthenized with pentobarbital 150mg/Kg IV. This and other procedures for raising polyclonal antibodiesare disclosed in E. Harlow, et. al., editors, Antibodies: A LaboratoryManual (1988), which is hereby incorporated by reference.

In addition to utilizing whole antibodies, the processes of the presentinvention encompass use of binding portions of such antibodies. Suchbinding portions include Fab fragments, F(ab′)₂ fragments, and Fvfragments. These antibody fragments can be made by conventionalprocedures, such as proteolytic fragmentation procedures, as describedin J. Goding, Monoclonal Antibodies: Principles and Practice, pp. 98-118(N.Y. Academic Press 1983), which is hereby incorporated by reference.

Alternatively, the processes of the present invention can utilize probesor ligands found either in nature or prepared synthetically byrecombinant DNA procedures or other biological or molecular procedures.Suitable probes or ligands are molecules which bind to the extracellulardomains of prostate specific membrane antigens identified by themonoclonal antibodies of the present invention. Other suitable probes orligands are molecules which bind to and are internalized with prostatespecific membrane antigens. Such probes or ligands can be, for example,proteins, peptides, lectins, or nucleic acid probes.

It is particularly preferred to use the monoclonal antibodies identifiedbelow in Table 1.

TABLE 1 ATCC Designation for Monoclonal Antibody Name Hybridoma CellLine E99 HB-12101 J415 HB-12109 J533 HB-12127 J591 HB-12126These antibodies can be used alone or as a component in a mixture withother antibodies or other biological agents to treat or image prostateepithelial cells with varying surface antigen characteristics.

Regardless of whether the biological agents are used for treatment ortherapy, they can be administered orally, parenterally, subcutaneously,intravenously, intramuscularly, intraperitoneally, by intranasalinstillation, by intracavitary or intravesical instillation,intraocularly, intraarterially, intralesionally, or by application tomucous membranes, such as, that of the nose, throat, and bronchialtubes. They may be administered alone or with pharmaceutically orphysiologically acceptable carriers, excipients, or stabilizers, and canbe in solid or liquid form such as, tablets, capsules, powders,solutions, suspensions, or emulsions.

The solid unit dosage forms can be of the conventional type. The solidform can be a capsule, such as an ordinary gelatin type containing thebiological agent, such as an antibody or binding portion thereof, of thepresent invention and a carrier, for example, lubricants and inertfillers such as, lactose, sucrose, or cornstarch. In another embodiment,these compounds are tableted with conventional tablet bases such aslactose, sucrose, or cornstarch in combination with binders like acacia,cornstarch, or gelatin, disintegrating agents such as, cornstarch,potato starch, or alginic acid, and a lubricant like stearic acid ormagnesium stearate.

The biological agent of the present invention may also be administeredin injectable dosages by solution or suspension of these materials in aphysiologically acceptable diluent with a pharmaceutical carrier. Suchcarriers include sterile liquids such as water and oils, with or withoutthe addition of a surfactant and other pharmaceutically andphysiologically acceptable carrier, including adjuvants, excipients orstabilizers. Illustrative oils are those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil, ormineral oil. In general, water, saline, aqueous dextrose and relatedsugar solution, and glycols such as, propylene glycol or polyethyleneglycol, are preferred liquid carriers, particularly for injectablesolutions.

For use as aerosols, the biological agent of the present invention insolution or suspension may be packaged in a pressurized aerosolcontainer together with suitable propellants, for example, hydrocarbonpropellants like propane, butane, or isobutane with conventionaladjuvants. The materials of the present invention also may beadministered in a non-pressurized form such as in a nebulizer oratomizer.

The biological agents may be utilized to detect normal, benignhyperplastic, and cancerous prostate epithelial cells in vivo. This isachieved by labeling the biological agent, administering the labeledbiological agent to a mammal, and then imaging the mammal.

Examples of labels useful for diagnostic imaging in accordance with thepresent invention are radiolabels such as ¹³¹I, ¹¹¹In, ¹²³I, ⁹⁹mTc, ³²P,¹²⁵I, ³H, ¹⁴C, and ¹⁸⁸Rh, fluorescent labels such as fluorescein andrhodamine, nuclear magnetic resonance active labels, positron emittingisotopes detectable by a positron emission tomography (“PET”) scanner,chemiluminescers such as luciferin, and enzymatic markers such asperoxidase or phosphatase. Short-range radiation emitters, such asisotopes detectable by short-range detector probes, such as atransrectal probe, can also be employed. These isotopes and transrectaldetector probes, when used in combination, are especially useful indetecting prostatic fossa recurrences and pelvic nodal disease. Thebiological agent can be labeled with such reagents using techniquesknown in the art. For example, see Wensel and Meares, Radioimmunoimagingand Radioimmunotherapy, Elsevier, New York (1983), which is herebyincorporated by reference, for techniques relating to the radiolabelingof antibodies. See also, D. Colcher et al., “Use of MonoclonalAntibodies as Radiopharmaceuticals for the Localization of HumanCarcinoma Xenografts in Athymic Mice”, Meth. Enzymol. 121: 802-816(1986), which is hereby incorporated by reference.

A radiolabeled biological agent of this invention can be used for invitro diagnostic tests. The specific activity of a tagged biologicalagent, such as a tagged antibody, binding portion thereof, probe, orligand, depends upon the half-life, the isotopic purity of theradioactive label, and how the label is incorporated into the biologicalagent. Table 2 lists several commonly-used isotopes, their specificactivities and half-lives. In immunoassay tests, the higher the specificactivity, in general, the better the sensitivity.

TABLE 2 Specific Activity of Pure Isotope Isotope (Curies/mole)Half-Life ¹⁴C 6.25 × 10¹  5720 years ³H 2.01 × 10⁴  12.5 years ³⁵S 1.50× 10⁶   87 days ¹²⁵I 2.18 × 10⁶   60 days ³²P 3.16 × 10⁶  14.3 days ¹³¹I1.62 × 10⁷  8.1 days

Procedures for labeling biological agents with the radioactive isotopeslisted in Table 2 are generally known in the art. Tritium labelingprocedures are described in U.S. Pat. No. 4,302,438, which is herebyincorporated by reference. Iodinating, tritium labeling, and ³⁵Slabeling procedures especially adapted for murine monoclonal antibodiesare described by Goding, J. W. (supra, pp 124-126) and the referencescited therein, which are hereby incorporated by reference. Otherprocedures for iodinating biological agents, such as antibodies, bindingportions thereof, probes, or ligands, are described by Hunter andGreenwood, Nature 144:945 (1962), David et al., Biochemistry13:1014-1021 (1974), and U.S. Pat. Nos. 3,867,517 and 4,376,110, whichare hereby incorporated by reference. Radiolabeling elements which areuseful in imaging include ¹²³I, ¹³¹I, ¹¹¹In, and ^(99m)Tc, for example.Procedures for iodinating biological agents are described by Greenwood,F. et al., Biochem. J. 89:114-123 (1963); Marchalonis, J., Biochem. J.113:299-305 (1969); and Morrison, M. et al., Immunochemistry, 289-297(1971), which are hereby incorporated by reference. Procedures for^(99m)Tc-labeling are described by Rhodes, B. et al. in Burchiel, S. etal. (eds.), Tumor Imaging: The Radioimmunochemical Detection of Cancer,New York: Masson 111-123 (1982) and the references cited therein, whichare hereby incorporated by reference. Procedures suitable for¹¹¹In-labeling biological agents are described by Hnatowich, D. J. etal., J. Immul. Methods, 65:147-157 (1983), Hnatowich, D. et al., J.Applied Radiation, 35:554-557 (1984), and Buckley, R. G. et al.,F.E.B.S. 166:202-204 (1984), which are hereby incorporated by reference.

In the case of a radiolabeled biological agent, the biological agent isadministered to the patient, is localized to the tumor bearing theantigen with which the biological agent reacts, and is detected or“imaged” in vivo using known techniques such as radionuclear scanningusing e.g., a gamma camera or emission tomography. See e.g., A. R.Bradwell et al., “Developments in Antibody Imaging”, MonoclonalAntibodies for Cancer Detection and Therapy, R. W. Baldwin et al.,(eds.), pp. 65-85 (Academic Press 1985), which is hereby incorporated byreference. Alternatively, a positron emission transaxial tomographyscanner, such as designated Pet VI located at Brookhaven NationalLaboratory, can be used where the radiolabel emits positrons (e.g., ¹¹C,¹⁸F, ¹⁵O, and ¹³N).

Fluorophore and chromophore labeled biological agents can be preparedfrom standard moieties known in the art. Since antibodies and otherproteins absorb light having wavelengths up to about 310 nm, thefluorescent moieties should be selected to have substantial absorptionat wavelengths above 310 nm and preferably above 400 nm. A variety ofsuitable fluorescers and chromophores are described by Stryer, Science,162:526 (1968) and Brand, L. et al., Annual Review of Biochemistry,41:843-868 (1972), which are hereby incorporated by reference. Thebiological agents can be labeled with fluorescent chromophore groups byconventional procedures such as those disclosed in U.S. Pat. Nos.3,940,475, 4,289,747, and 4,376,110, which are hereby incorporated byreference.

One group of fluorescers having a number of the desirable propertiesdescribed above are the xanthene dyes, which include the fluoresceinsderived from 3,6-dihydroxy-9-henylxanthhydrol and resamines andrhodamines derived from 3,6-diamino-9-phenylxanthydrol and lissanimerhodamine B. The rhodamine and fluorescein derivatives of9-o-carboxyphenylxanthhydrol have a 9-o-carboxyphenyl group. Fluoresceincompounds having reactive coupling groups such as amino andisothiocyanate groups such as fluorescein isothiocyanate andfluorescamine are readily available. Another group of fluorescentcompounds are the naphthylamines, having an amino group in the α or βposition.

Biological agents can be labeled with fluorchromes or chromophores bythe procedures described by Goding, J. (supra, pp 208-249). Thebiological agents can be labeled with an indicating group containing theNMR-active ¹⁹F atom, or a plurality of such atoms inasmuch as (i)substantially all of naturally abundant fluorine atoms are the ¹⁹Fisotope and, thus, substantially all fluorine-containing compounds areNMR-active; (ii) many chemically active polyfluorinated compounds suchas trifluoracetic anhydride are commercially available at relatively lowcost, and (iii) many fluorinated compounds have been found medicallyacceptable for use in humans such as the perfluorinated polyethersutilized to carry oxygen as hemoglobin replacements. After permittingsuch time for incubation, a whole body NMR determination is carried outusing an apparatus such as one of those described by Pykett, ScientificAmerican, 246:78-88 (1982), which is hereby incorporated by reference,to locate and image prostate epithelial cells.

In cases where it is important to distinguish between regions containinglive and dead prostate epithelial cells or to distinguish between liveand dead prostate epithelial cells, the antibodies of the presentinvention (or other biological agents of the present invention), labeledas described above, can be coadministered along with an antibody orother biological agent which recognizes only living or only deadprostate epithelial cells labeled with a label which can bedistinguished from the label used to label the subject antibody. Bymonitoring the concentration of the two labels at various locations ortimes, spatial and temporal concentration variations of living and deadnormal, benign hyperplastic, and cancerous prostate epithelial cells canbe ascertained. In particular, this method can be carried out using thelabeled antibodies of the present invention, which recognize both livingand dead epithelial prostate cells, and labeled 7E11 antibodies, whichrecognize only dead epithelial prostate cells.

The biological agents can also be utilized to kill or ablate normal,benign hyperplastic, and cancerous prostate epithelial cells in vivo.This involves using the biological agents by themselves or with acytotoxic drug to which the biological agents recognizing normal, benignhyperplastic, and cancerous prostate epithelial cells are bound. Thisinvolves administering the biological agents bonded to a cytotoxic drugto a mammal requiring such treatment. Since the biological agentsrecognize prostate epithelial cells, any such cells to which thebiological agents bind are destroyed. Although such administration maydestroy normal prostate epithelial cells, this is not problematic,because the prostate is not required for life or survival. Although theprostate may indirectly contribute to fertility, this is not likely tobe a practical consideration in patients receiving the treatment of thepresent invention.

The biological agents of the present invention may be used to deliver avariety of cytotoxic drugs including therapeutic drugs, a compoundemitting radiation, molecules of plants, fungal, or bacterial origin,biological proteins, and mixtures thereof. The cytotoxic drugs can beintracellularly acting cytotoxic drugs, such as short-range radiationemitters, including, for example, short-range, high-energy α-emitters.

Enzymatically active toxins and fragments thereof are exemplified bydiphtheria toxin A fragment, nonbinding active fragments of diphtheriatoxin, exotoxin A (from Pseudomonas aeruginosa), ricin A chain, abrin Achain, modeccin A chain, α-sacrin, certain Aleurites fordii proteins,certain Dianthin proteins, Phytolacca americana proteins (PAP, PAPII andPAP-S), Morodica charantia inhibitor, curcin, crotin, Saponariaofficinalis inhibitor, gelonin, mitogillin, restrictocin, phenomycin,and enomycin, for example. Procedures for preparing enzymatically activepolypeptides of the immunotoxins are described in WO84/03508 andWO85/03508, which are hereby incorporated by reference. Certaincytotoxic moieties are derived from adriamycin, chlorambucil,daunomycin, methotrexate, neocarzinostatin, and platinum, for example.

Procedures for conjugating the biological agents with the cytotoxicagents have been previously described. Procedures for conjugatingchlorambucil with antibodies are described by Flechner, I, EuropeanJournal of Cancer, 9:741-745 (1973); Ghose, T. et al., British MedicalJournal, 3:495-499 (1972); and Szekerke, M., et al., Neoplasma,19:211-215 (1972), which are hereby incorporated by reference.Procedures for conjugating daunomycin and adriamycin to antibodies aredescribed by Hurwitz, E. et al., Cancer Research, 35:1175-1181 (1975)and Amon, R. et al. Cancer Surveys, 1:429-449 (1982), which are herebyincorporated by reference. Procedures for preparing antibody-ricinconjugates are described in U.S. Pat. No. 4,414,148 and by Osawa, T., etal. Cancer Surveys, 1:373-388 (1982) and the references cited therein,which are hereby incorporated by reference. Coupling procedures as alsodescribed in EP 86309516.2, which is hereby incorporated by reference.

In a particularly preferred embodiment of the present invention, a firstbiological agent is conjugated with a prodrug which is activated onlywhen in close proximity with a prodrug activator. The prodrug activatoris conjugated with a second biological agent according to the presentinvention, preferably one which binds to a non-competing site on theprostate specific membrane antigen molecule. Whether two biologicalagents bind to competing or non-competing binding sites can bedetermined by conventional competitive binding assays. For example,monoclonal antibodies J591, J533, and E99 bind to competing bindingsites on the prostate specific membrane antigen molecule. Monoclonalantibody J415, on the other hand, binds to a binding site which isnon-competing with the site to which J591, J533, and E99 bind. Thus, forexample, the first biological agent can be one of J591, J533, and E99,and the second biological agent can be J415. Alternatively, the firstbiological agent can be J415, and the second biological agent can be oneof J591, J533, and E99. Drug-prodrug pairs suitable for use in thepractice of the present invention are described in Blakely et al.,“ZD2767, an Improved System for Antibody-directed Enzyme Prodrug TherapyThat Results in Tumor Regressions in Colorectal Tumor Xenografts,”Cancer Research, 56:3287-3292 (1996), which is hereby incorporated byreference.

Alternatively, the biological agent can be coupled to high energyradiation emitters, for example, a radioisotope, such as ¹³¹I, aγ-emitter, which, when localized at the tumor site, results in a killingof several cell diameters. See, e.g., S. E. Order, “Analysis, Results,and Future Prospective of the Therapeutic Use of Radiolabeled Antibodyin Cancer Therapy”, Monoclonal Antibodies for Cancer Detection andTherapy, R. W. Baldwin et al. (eds.), pp 303-316 (Academic Press 1985),which is hereby incorporated by reference. Other suitable radioisotopesinclude α-emitters, such as ²¹²Bi, ²¹³Bi, and ²¹¹At, and β-emitters,such as ¹⁸⁶Re and ⁹⁰Y. Radiotherapy is expected to be particularlyeffective, because prostate cancer is a relatively radiosensitive tumor.

Where the biological agents are used alone to kill or ablate prostateepithelial cells, such killing or ablation can be effected by initiatingendogenous host immune functions, such as complement-mediated orantibody-dependent cellular cytotoxicity.

The biological agent of the present invention can be used and soldtogether with equipment, as a kit, to detect the particular label.

Biological agents of the present invention can be used in conjunctionwith other therapeutic treatment modalities. Such other treatmentsinclude surgery, radiation, cryosurgery, thermotherapy, hormonetreatment, chemotherapy, vaccines, and other immunotherapies.

Also encompassed by the present invention is a method of killing orablating which involves using the biological agents for prophylaxis. Forexample, these materials can be used to prevent or delay development orprogression of prostate cancer.

Use of the prostate cancer therapy of the present invention has a numberof benefits. Since the biological agents according to the presentinvention only target prostate epithelial cells, other tissue is spared.As a result, treatment with such biological agents is safer,particularly for elderly patients. Treatment according to the presentinvention is expected to be particularly effective, because it directshigh levels of biological agents, such as antibodies or binding portionsthereof, probes, or ligands, to the bone marrow and lymph nodes whereprostate cancer metastases predominate. Moreover, tumor sites forprostate cancer tend to be small in size and, therefore, easilydestroyed by cytotoxic agents. Treatment in accordance with the presentinvention can be effectively monitored with clinical parameters such asserum prostate specific antigen and/or pathological features of apatient's cancer, including stage, Gleason score, extracapsular,seminal, vesicle or perineural invasion, positive margins, involvedlymph nodes, etc. Alternatively, these parameters can be used toindicate when such treatment should be employed.

Because the biological agents of the present invention bind to livingprostate cells, therapeutic methods using these biological agents aremuch more effective than those which target lysed prostate cells. Forthe same reasons, diagnostic and imaging methods which determine thelocation of living normal, benign hyperplastic, or cancerous prostateepithelial cells are much improved by employing the biological agents ofthe present invention. In addition, the ability to differentiate betweenliving and dead prostate cells can be advantageous, especially tomonitor the effectiveness of a particular treatment regimen.

Hybridomas E99, J415, J533, and J591 have been deposited pursuant to,and in satisfaction of, the requirements of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure with the American Type Culture Collection(“A.T.C.C.”) at 10801 University Boulevard, Manassas, Va. 30110-2209.Hybridoma E99 was deposited on May 2, 1996, and received A.T.C.C.Designation Number HB-12101. Hybridoma J415 was deposited on May 30,1996, and received A.T.C.C. Designation Number HB-12109. Hybridomas J533and J591 were deposited on Jun. 6, 1996, and received A.T.C.C.Designation Numbers HB-12127 and HB-12126, respectively.

The present invention is further illustrated by the following examples.

EXAMPLES Example 1 Human Tissues

Fresh specimens of benign and malignant tissues were obtained from theDepartment of Pathology of New York Hospital Cornell University MedicalCenter (“NYH-CUMC”),

Example 2 Tissue Culture

Cultured cell lines of human cancers were obtained from the Laboratoryof Urological Oncology of NYH-CUMC. The prostate cancer cell lines PC-3(Mickey, D. D., et al., “Characterization Of A Human ProstateAdenocarcinoma Cell Line (DU145) As A Monolayer Culture And As A SolidTumor In Athymic Mice,” Prog. Clin. Biol. Res., 37:67-84 (1980), whichis hereby incorporated by reference), DU-145 (Mickey, D. D., et al.,“Characterization Of A Human Prostate Adenocarcinoma Cell Line (DU145)As A Monolayer Culture And As A Solid Tumor In Athymic Mice,” Prog.Clin. Biol. Res., 37:67-84 (1980), which is hereby incorporated byreference), and LNCaP (Horoszewicz, J. S., et al., “LNCaP Model Of HumanProstatic Carcinoma,” Cancer Res., 43: 1809-1818 (1983), which is herebyincorporated by reference) were obtained from the American Type CultureCollection (Rockville, Md.). Hybridomas were initially cloned inRPMI-1640 medium supplemented with 10% FCS, 0.1 mM nonessential aminoacids, 2 mM L-glutamine, 100 units/ml of penicillin, 100 ug/ml ofstreptomycin and HAT medium (GIBCO, Grand Island, N.Y.). Subclones werecultured in the same medium without aminopterin.

Example 3 Preparation of Mouse Monoclonal Antibodies

Female BALB/c mice were immunized intraperitoneally with LNCaP (6×10⁶cells) three times at 2 week intervals. A final intraperitoneal boosterimmunization was administered with fresh prostate epithelial cells whichhad been grown in vitro. Three days later, spleen cells were fused withSP-2 mouse myeloma cells utilizing standard techniques (Ueda, R., etal., “Cell Surface Antigens Of Human Renal Cancer Defined By MouseMonoclonal Antibodies: Identification Of Tissue-Specific KidneyGlycoproteins,” Proc. Natl. Acad. Sci. USA, 78:5122-5126 (1981), whichis hereby incorporated by reference). Supernatants of the resultingclones were screened by rosette and complement cytotoxicity assaysagainst viable LNCaP. Clones which were positive by these assays werescreened by immunochemistry vs normal kidney, colon, and prostate.Clones which were LNCap⁺/NmlKid⁻/colon⁻/prostate⁺ were selected andsubcloned 3 times by limiting dilution. The immunoglobulin class ofcultured supernatant from each clone was determined by immunodiffusionusing specified rabbit antisera (Calbiochem, San Diego, Calif.). mAbswere purified using the MAPS-II kit (Bio-Rad, Richmond, Calif.)

Example 4 Biotinylation of mAbs

Purified mAbs were dialyzed in 0.1 M NaHCO₃ for 2 hours. One ml of mAbat 1 mg/ml was mixed with 0.1 ml of biotinamidocaproateN-hydroxysuccinamide ester (Sigma) in dimethylsulfoxide (1 mg/ml) andstirred for 4 hours at room temperature. Unbound biotin was removed bydialysis against phosphate buffered saline (“PBS”).

Example 5 Immunohistochemical Staining

Cryostat sections of prostate tissues were placed inside rings of Falcon3034 plate covers (Becton-Dickenson, Lincoln Park, N.J.) previouslycoated with 0.45% gelatin solution as described in Marusich, M. F., “ARapid Method For Processing Very Large Numbers Of Tissue Sections ForImmunohistochemical Hybridoma Screening,” J. Immunol. Methods,111:143-145 (1988), which is hereby incorporated by reference. Plateswere stored at −80° C. Cryostat sections were fixed with 2%paraformaldehyde in PBS for 10 min at room temperature, and, afterwashing with PBS, endogenous peroxidase activity was blocked bytreatment with 0.3% hydrogen peroxide in PBS for 10 min at roomtemperature. After sections were incubated with 2% BSA in PBS for 20min, mAbs were added for 60 min at room temperature. Slides wereextensively washed with PBS and incubated with peroxidase-conjugatedrabbit anti-mouse Ig (DAKO Corp., Santa Barbara, Calif.) diluted 1:100in 10% normal human serum in PBS for 60 min at room temperature. After adiaminobenzidine reaction, sections were counterstained withhematoxylin.

Example 6 Serological Analysis

The anti-mouse immunoglobulin mixed hemadsorption assay was performed asdescribed in Ueda, R., et al., “Cell Surface Antigens Of Human RenalCancer Defined By Mouse Monoclonal Antibodies: Identification OfTissue-Specific Kidney Glycoproteins,” Proc. Natl. Acad. Sci. USA,78:5122-5126 (1981), which is hereby incorporated by reference. Toprepare the indicator cells, anti-mouse Ig (DAKO Corp.) was conjugatedto type O human RBC using 0.01% chromium chloride. Serological assayswere performed on cells previously plated in Terasaki plates (Nunc,Denmark). Antibodies were incubated with target cells at roomtemperature for 1 hour. Target cells were then washed, and indicatorcells added for 1 hour.

Example 7 Immunoprecipitation

LNCaP cells (2×10⁷) were biotinylated with biotin-NHSS (at finalconcentration of 5 mM) for 30 minutes on ice. After washing, thebiotinylated cells were resuspended in 1 ml lysis buffer (20 mM Tris/HClpH 8.0, 1 mM EDTA, 1 mM PMSF, 1% triton X-100) for 30 min on ice. Thesuspension was centrifuged at 1500 g×100 min at 4° C., and thesupernatant was centrifuged at 12,000 rpm×15 min at 4° C. The resultinglysate was preabsorbed with rabbit or goat anti-mouse IgG-coatedpansorbin for 1 hour at 4° C. The pre-absorbed lysate was incubated withthe mAb overnight at 4° C. Rabbit or goat anti-mouse Ig-coated agarosebeads were added for 2 hours at 4° C. and then washed. The beads wereresuspended in Tris-base/NaCl, added to sample buffer with2-mercaptoethanol, and boiled for 5 min. After centrifuging, thesupernatant was run on an SDS-PAGE 12% gel. The gel was transferred to anitrocellulose membrane which was blocked and stained withstraptavidin-peroxidase. The membrane was developed withdiaminobenzidine (“DAB”).

Sequential immunoprecipitation was similar except that the lysate wasinitially pre-cleared with one mAb overnight at 4° C. A second mAb wasthen used to immunoprecipitate the pre-cleared lysate.

Approximately 2000 clones were screened, of which four clones wereselected as described in Example 3, above. After subcloning,supernatants from the 4 hybridomas, E99, J415, J533, and J591, wereassayed by immunofluorescence against viable (i.e. unfixed) LNCaP,immunoprecipitation, and sequential immunoprecipitation to confirmreactivity to PSMA.

The immunofluorescence study using the LNCaP target cell (describedoriginally in Horoszewicz, which is hereby incorporated by reference, tomake the 7E11 antibody and the prototype cell line for expression forPSMA) shows that E99 antibody binds to and renders viable LNCaP cellsimmunofluorescent. This is in contrast to the 7E11 antibody, which, asnoted originally in Horoszewicz, which is hereby incorporated byreference, gives only poor or no binding to viable LNCaP cells butexhibits strong binding once the cells are fixed (killed).

The reactivities of the four mAbs with normal human tissues wereexamined immunohistochemically; these results are presented in Table 3.

TABLE 3 Reactivity of mAbs with human normal tissues by indirectimmunoperosidase staining E99 J415 J533 J591 Tissues (γ₃) (γ₁) (γ₁) (γ₁)Prostate* • • • • Kidney Glomerulus ∘ ∘ ∘ ∘ Prox. Tubule ▪ ▪ ▪ ▪ Ureter∘ ∘ ∘ ∘ Bladder ∘ ∘ ∘ ∘ Testis ∘ ∘ ∘ ∘ Uterus ∘ Esophagus ∘ ∘ ∘ ∘ SmallIntestine ∘ ∘ ∘ ∘ Stomach ∘ ∘ ∘ ∘ Colon ∘ ∘ ∘ ∘ Spleen ∘ ∘ ∘ ∘ Thyroid ∘∘ ∘ ∘ Lung ∘ ∘ ∘ ∘ Pancreas ∘ ∘ ∘ ∘ Liver ∘ ∘ ∘ ∘ *BPH 0-3⁺ 0-3⁺ 0-4⁺0-4⁺ *Prostate Cancer 0-3⁺ 0-3⁺ 0-4⁺ 0-4⁺ *LNCaP (scid) 3⁺ 3⁺ 4⁺ 4⁺*LuCaP (scid) 0-2⁺ 0-2⁺ 0-3⁺ 0-3⁺ • - positive; ▪ - weak, heterogeneous;∘ - negativeThe above sequential immunoprecipitaion study showed that 7E11, E99,J415, J533, and J591 bind to the same molecule, i.e. PSMA.

Example 8 Western Blot Analysis

To confirm that antibodies E99, J415, J533, and J591 precipitate anidentical band to the 7E11 antibody (i.e., PSMA), Western Blot analyseswere performed. Seminal plasma (400 μg/lane) or LNCaP lysate were loadedinto lanes of 12% SDS-PAGE gels. After electrophoresis, the gels aretransferred to nitrocellulose membranes. The membranes were blocked with51 dry milk/Tris-buffered saline-tween 20 (“TBST”) for 60 min at roomtemperature. After washing, the membranes were incubated with primarymAb for 60 min at room temperature. After repeat washing, the membraneswere incubated with sheep anti-mouse-Ig-peroxidase 1/5000 in 5% drymilk/TBST for 60 min at room temperature. After repeat washing, themembranes were developed using a chemiluminescent tag designated “ECL”(Amersham Life Sciences, International, Arlington Heights, Ill.)according to the manufacturer's directions. The results of the WesternBlot experiment are presented in Table 4.

TABLE 4 Western blot data Sample 7E11 E99 J415 J533 J591 Prostatic 100KD 100 KD 100 KD 100 KD 100 KD (seminal) band band band band band fluidLNCaP 100 KD & 100 KD & 100 KD & 100 KD & 100 KD & cell lysate 200 KD200 KD 200 KD 200 KD 200 KD bands bands bands bands bands

Example 9 mAb Reactivity to External Domain of PSMA

To confirm cell surface (external) expression of the detected PSMA,fresh, viable LNCaP cells were tested, without fixation, in vitro, byimmunofluorescence. LNCaP cells were washed and incubated with mAb for 1hour at room temperature and then with a rabbit anti-mouseIg-fluorescein (DAKO Corp., Santa Barbara, Calif.). Wells were read witha fluorescent microscope. Negative control consisted of anisotype-matched irrelevant mAb, while an anti-class I MHC mAb served asa positive control.

Immunofluorescence and rosette assay results are presented in Table 5.

TABLE 5 Comparison of 7E11 with new mAbs LNCaP viable cells 7E11 E99J415 J533 J591 Immunofluorescence neg 3+ 3+ 4+ 4+ Rosette neg + + + +assay LNCaP-fixed +++ ++++ +++ ++ +++

Example 10 Competition Studies

A competition study was carried out to determine whether J591, J533,E99, and J415 detected the same or different antigenic sites (epitopes)of the prostate specific membrane antigen molecule using the followingprocedure.

Plates were coated with LNCaP cell line lysate as a source of prostatespecific membrane antigen and washed to remove unbound material. “Cold”(unlabeled) monoclonal antibody was incubated on the plate for 1 hour atroom temperature to allow binding to its antigenic site. Subsequently, asecond monoclonal antibody, labeled either with biotin or ¹²⁵I, wasadded for an additional hour. Plates were washed to remove unboundmaterial. The amount of the second monoclonal antibody bound to theprostate specific membrane antigen-coated plate was determined either byavidin-alkaline phosphatase in an enzyme-linked immunoassay (in the caseof biotin-labeled second monoclonal antibody) or by physically countingthe well in a gamma counter (in the case of ¹²⁵I-labeled secondmonoclonal antibody). Controls consisted of using the same monoclonalantibody both cold and labeled to define “100% competition” or usingmonoclonal antibody to a totally different molecule (e.g., monoclonalantibody I-56, which detects inhibin, a prostate related proteindifferent from prostate specific membrane antigen) to define “0%competition”.

The results indicated that J591, J533, and E99 each interfere, compete,or block binding of one another but do not block binding of J415 andvice versa. 7E11/CYT356, known to bind PSMA at a different(intracellular) site, did not block any of J591, J533, E99, or J415.

Having pairs of monoclonal antibodies which bind to non-competing sitespermits development of antibody sandwich assays for detecting solubleantigens, such as solubilized prostate specific membrane antigen orfragment thereof, in, for example, body fluids. For example, the antigen(e.g., prostate specific membrane antigen or a fragment thereof) couldbe “captured” from body fluid with J591 and, in another step, detectedby labeled J415.

In another setting, e.g. treatment, one could increase antibody bindingby using a combination of non-competing monoclonal antibodies. Forexample, assuming the non-competing sites are each represented once onthe prostate specific membrane antigen molecule, adding a combination ofJ591 plus J415 would bind twice as many monoclonal antibody molecules aseither monoclonal antibody alone. Binding two non-competing antigenicbinding sites also can result in greater antigen cross-linking and,perhaps, increased internalization. Furthermore, since the two detectedsites are physically located on the same prostate specific membraneantigen molecule, the binding of two monoclonal antibody molecules tothat single prostate specific membrane antigen molecule puts the twomonoclonal antibody molecules in close proximity to each other, asetting which provides optimal drug-prodrug interaction. For example,monoclonal antibody J591 can be conjugated with an inactive pro-drug andJ415 can be conjugated with a pro-drug activator. Since prodrug andactivator would be bound in close proximity only at the site of prostatespecific membrane antigen-expressing cells (e.g., prostate cancercells), prodrug activation to the active form would occur only at thosesites.

Example 11 Microscopy

Confocal microscopy and immuno-electron microscopy demonstrated thatE99, J591, J533, and J415 are bound to the cell membrane atclathrin-coated pits and then rapidly internalize into endosomes(cytoplasmic vesicles). FIGS. 1-4 are immuno-electron micrographs whichfollow the interaction of gold-labeled monoclonal antibody J591 with thecell surface as a function of time. In these figures, the location ofthe monoclonal antibody is indicated by the black dots.

Viable LNCaP cells were incubated with J591 for one hour at 4° C. Thecells were washed and then held at 37° C. for 0, 5, 10, or 15 minutes,after which time they were fixed and processed for immuno-electronmicroscopy. FIG. 1 shows the cell prior to 37° C. incubation. J591 canbe seen bound to the cell along the external aspect of the cellmembrane. In this Figure, “M” denotes the cell's mitochondria, and “N”denotes its nucleus. FIG. 2 shows the cell after incubation at 37° C.for 5 minutes. The arrow indicates formation of a clathrin-coated pit.In FIG. 3, which shows the cell after a 10 minute 37° C. incubation,pinching off or endocytosis of the clathrin-coated pit can be seen, asindicated by the arrow. FIG. 4 shows that, after incubation at 37° C.for 15 minutes, monoclonal antibody J591 is contained in endocyticvesicles within the cell, as indicated by the arrows. As can be seen inFIG. 5, after incubation at 37° C. for 15 minutes, monoclonal antibodyJ591 is also contained within endosomes, as indicated by the arrows.

Example 12 Sequencing of the Variable Region of Monoclonal Antibody J591

Total RNA was prepared from 10⁷ murine hybridoma J591 cells. A sample ofthe conditioned medium from these cells was tested for binding to thespecific antigen for J591 on prostate cells. The conditioned medium waspositive by both ELISA and Western Blot for binding to the antigen.

VH and VK cDNA were prepared using reverse transcriptase and mouse κconstant region and mouse IgG constant region primers. The first strandcDNAs were amplified by PCR using a variety of mouse signal sequenceprimers (6 for VH and 7 for VK). The amplified DNAs were gel-purifiedand cloned into the vector pT7Blue.

The VH and VK clones obtained were screened for correct inserts by PCR,and the DNA sequence of selected clones was determined by the dideoxychain termination method.

Excluding the primer region (as the sequence of this depended on thesequence of the primer that was used), all the VH clones obtained gaveidentical sequence. This sequence was obtained from clones produced withthree different 5′ primers. One clone had one base pair change withinthe signal sequence, and one clone contained an aberrant PCR product.Using the sequencing strategy shown in FIG. 6, the nucleotide sequencefor the heavy chain was obtained. It is designated SEQ. ID. No. 1 and ispresented in FIG. 7, along with the nucleotide sequence of thecorresponding reverse, non-coding strand (designated SEQ. ID. No. 2).These sequences include part of the signal sequence and part of theconstant region of the antibody. The corresponding deduced amino acidsequences of J591 VH, designated SEQ. ID. No. 3, SEQ. ID. No. 4, andSEQ. ID. No. 5, are also shown in FIG. 7. The coding strand of the J591heavy chain's variable region (exclusive of signal sequence and constantregion components) has the following nucleotide sequence (designatedSEQ. ID. No. 6):

GAGGTCCAGCTGCAACAGTCTGGACCTGAACTGGTGAAGCCTGGGACTTCAGTGAGGATATCCTGCAAGACTTCTGGATACACATTCACTGAATATACCATACACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAAACATCAATCCTAACAATGGTGGTACCACCTACAATCAGAAGTTCGAGGACAAGGCCACATTGACTGTAGACAAGTCCTCCAGTACAGCCTACATGGAGCTCCGCAGCCTAACATCTGAGGATTCTGCAGTCTATTATTGTGCAGCTGGTTGGAACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCT CAThe reverse, non-coding strand of the JS91 heavy chain's variable region(exclusive of signal sequence and constant region components) has thefollowing nucleotide sequence (designated SEQ. ID. No. 7):

TGAGGAGACTGTGAGAGTGGTGCCTTGGCCCCAGTAGTCAAAGTTCCAACCAGCTGCACAATAATAGACTGCAGAATCCTCAGATGTTAGGCTGCGGAGCTCCATGTAGGCTGTACTGGAGGACTTGTCTACAGTCAATGTGGCCTTGTCCTCGAACTTCTGATTGTAGGTGGTACCACCATTGTTAGGATTGATGTTTCCAATCCACTCAAGGCTCTTTCCATGGCTCTGCTTCACCCAGTGTATGGTATATTCAGTGAATGTGTATCCAGAAGTCTTGCAGGATATCCTCACTGAAGTCCCAGGCTTCACCAGTTCAGGTCCAGACTGTTGCAGCTGGACC TC

The protein sequence corresponding to the J591 heavy chain's variableregion (exclusive of signal sequence and constant region components) hasthe following amino acid sequence (designated SEQ ID NO. 8):

EVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAA GWNFDYWGQGTTLTVSS

The J591 VH is in Mouse Heavy Chains Subgroup IIA (designated SEQ ID No.20) (Kabat et al., Sequences of Proteins of Immunological Interest, U.S.Department of Health and Human Services (1991) (“Kabat”), which ishereby incorporated by reference). The sequence of J591 VH is comparedto the consensus sequence for this subgroup in FIG. 8.

In contrast to the VH, more than one VK sequence was obtained. Out ofthe 15 VK clones examined, four gave the sequence of an aberrant mouseIgκ from the fusion partner (Carol et al., Molecular Immunology,25:991-995 (1988), which is hereby incorporated by reference). Theseclones originated from two specific 5′ primers. No further work was donewith these clones. Of the remaining clones, ten gave identicalnucleotide sequences, and one clone, VK17, gave an alternative VKsequence. The ten identical clones originated from three 5′ primers(different from the two that gave the aberrant sequence), one of whichalso produced VK17. The sequencing strategy that was employed is shownin FIG. 9.

The nucleic acid sequence of J591 VK corresponding to the ten identicalclones (designated SEQ. ID. No. 9) is presented in FIG. 10, along withthe nucleic acid sequence of the corresponding reverse, non-codingstrand (designated SEQ. ID. No. 10) and the deduced amino acidsequences, which are designated SEQ. ID. No. 11, SEQ. ID. No. 12, andSEQ. ID. No. 13. These sequences include part of the signal sequence andpart of the constant region of the antibody. The coding strand of theJ591 light (kappa) chain's variable region (exclusive of signal sequenceand constant region components) corresponding to the ten identicalclones has the following nucleotide sequence (designated SEQ. ID. No.14):

AACATTGTAATGACCCAATCTCCCAAATCCATGTCCATGTCAGTAGGAGAGAGGGTCACCTTGACCTGCAAGGCCAGTGAGAATGTGGTTACTTATGTTTCCTGGTATCAACAGAAACCAGAGCAGTCTCCTAAACTGCTGATATACGGGGCATCCAACCGGTACACTGGGGTCCCCGATCGCTTCACAGGCAGTGGATCTGCAACAGATTTCACTCTGACCATCAGCAGTGTGCAGGCTGAAGACCTTGCAGATTATCACTGTGGACAGGGTTACAGCTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAThe reverse, non-coding strand of the J591 light (kappa) chain'svariable region (exclusive of signal sequence and constant regioncomponents) corresponding to the ten identical clones has the followingnucleotide sequence (designated SEQ. ID. No. 15):

TTTTATTTCCAGCTTGGTCCCCCCTCCGAACGTGTACGGATAGCTGTAACCCTGTCCACAGTGATAATCTGCAAGGTCTTCAGCCTGCACACTGCTGATGGTCAGAGTGAAATCTGTTGCAGATCCACTGCCTGTGAAGCGATCGGGGACCCCAGTGTACCGGTTGGATGCCCCGTATATCAGCAGTTTAGGAGACTGCTCTGGTTTCTGTTGATACCAGGAAACATAAGTAACCACATTCTCACTGGCCTTGCAGGTCAAGGTGACCCTCTCTCCTACTGACATGGACATGGATTTGGGAGATTGGGTCATTACAATGTT

The protein sequence corresponding to the J591 light (kappa) chain'svariable region (exclusive of signal sequence and constant regioncomponents) corresponding to ten identical clones has the followingamino acid sequence (designated SEQ ID NO. 16):

NIVMTQSPKSMSMSVGERVTLTCKASENVVTYVSWYQQKPEQSPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQGYSYPY TFGGGTKLEIK

The coding strand of the J591 light (kappa) chain's variable region(exclusive of signal sequence and constant region components)corresponding to clone VK17 has the following nucleotide sequence(designated SEQ. ID. No. 17):

GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCATCTGTAAGGCCAGTCAAGATGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTATTGGGCATCCACTCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGACTTCACTCTCACCATTACTAATGTTCAGTCTGAAGACTTGGCAGATTATTTCTGTCAGCAATATAACAGCTATCCTCTCACGTTCGGTGCTGGGACCATGCTGGACCTGAAAThe reverse, non-coding strand of the J591 light (kappa) chain'svariable region (exclusive of signal sequence and constant regioncomponents) corresponding to clone VK17 has the following nucleotidesequence (designated SEQ. ID. No. 18):

TTTCAGGTCCAGCATGGTCCCAGCACCGAACGTGAGAGGATAGCTGTTATATTGCTGACAGAAATAATCTGCCAAGTCTTCAGACTGAACATTAGTAATGGTGAGAGTGAAGTCTGTCCCAGATCCACTGCCTGTGAAGCGATCAGGGACTCCAGTGTGCCGAGTGGATGCCCAATAAATCAGTAGTTTAGGAGATTGTCCTGGTTTCTGTTGATACCAGTCTACAGCAGTACCCACATCTTGACTGGCCTTACAGATGATGCTGACCCTGTCTCCTACTGATGTGGACATGAATTTGTGAGACTGGGTCATCACAATGTC

The protein sequence corresponding to the J591 light (kappa) chain'svariable region (exclusive of signal sequence and constant regioncomponents) corresponding to clone VK17 has the following amino acidsequence (designated SEQ ID NO. 19):

DIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTF GAGTMLDLKJ591 VK is in the Mouse Kappa Chains Subgroup V (designated SEQ ID No.21) (Kabat, which is hereby incorporated by reference). The sequence ofJ591 VK corresponding to the ten identical clones is compared to theconsensus sequence for the subgroup in FIG. 11.

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose and variations can be made by those skilled in the art withoutdeparting from the spirit and scope of the invention which is defined bythe following claims.

1. A method of treating prostate cancer comprising: providing amonoclonal antibody or antigen binding portion thereof which binds toprostate specific membrane antigen (PSMA) and competes for binding toPSMA with a monoclonal antibody selected from the group consisting of amonoclonal antibody produced by a hybridoma with an ATCC accessionnumber HB-12101, a monoclonal antibody produced by a hybridoma with anATCC accession number HB-12127, and a monoclonal antibody produced by ahybridoma with an ATCC accession number HB-12126 wherein the antibody orantigen binding portion thereof is conjugated to a cytotoxic drug; andadministering the antibody or antigen binding portion thereof to asubject under conditions effective to treat prostate cancer.
 2. Themethod according to claim 1, wherein the prostate cancer is metastaticprostate cancer.
 3. The method according to claim 2, wherein themetastatic prostate cancer involves a bone marrow or a lymph nodemetastasis.
 4. The method according to claim 1, wherein theadministering is carried out parenterally.
 5. The method according toclaim 1, wherein the administering is carried out intravenously.
 6. Themethod according to claim 1, wherein the administering is carried out byintracavitary instillation.
 7. The method according to claim 1, whereinthe monoclonal antibody or antigen binding portion thereof isadministered following a prostatectomy.
 8. The method according to claim1, wherein the monoclonal antibody or antigen binding portion binds livecells.
 9. The method according to claim 1, wherein the monoclonalantibody provided is produced by a hybridoma with an ATCC accessionnumber HB-12101.
 10. A method of treating prostate cancer comprising:providing a monoclonal antibody or antigen binding portion thereof whichbinds to prostate specific membrane antigen (PSMA) and competes forbinding to PSMA with a monoclonal antibody selected from the groupconsisting of a monoclonal antibody produced by a hybridoma with an ATCCaccession number HB-12101, a monoclonal antibody produced by a hybridomawith an ATCC accession number HB-12127, and a monoclonal antibodyproduced by a hybridoma with an ATCC accession number HB-12126, whereinthe antibody is labeled with the radiolabel ⁹⁰Y; and administering theantibody or antigen binding portion thereof to a subject underconditions effective to treat prostate cancer.
 11. A method of treatingprostate cancer comprising: providing a monoclonal antibody or antigenbinding portion thereof which binds to prostate specific membraneantigen (PSMA) and competes for binding to PSMA with a monoclonalantibody selected from the group consisting of a monoclonal antibodyproduced by a hybridoma with an ATCC accession number HB-12101, amonoclonal antibody produced by a hybridoma with an ATCC accessionnumber HB-12127, and a monoclonal antibody produced by a hybridoma withan ATCC accession number HB-12126, wherein the antibody is labeled witha radiolabel, and wherein the radiolabel is a beta- or gamma-emitter;and administering the antibody or antigen binding portion thereof to asubject under conditions effective to treat prostate cancer.
 12. Amethod of treating prostate cancer comprising: providing a monoclonalantibody or antigen binding portion thereof which binds to prostatespecific membrane antigen (PSMA) and competes for binding to PSMA with amonoclonal antibody selected from the group consisting of a monoclonalantibody produced by a hybridoma with an ATCC accession number HB-12101,a monoclonal antibody produced by a hybridoma with an ATCC accessionnumber HB-12127, and a monoclonal antibody produced by a hybridoma withan ATCC accession number HB-12126, wherein the antibody is bound to acytotoxic drug of bacterial origin; and administering the antibody orantigen binding portion thereof to a subject under conditions effectiveto treat prostate cancer.
 13. A method of treating prostate cancercomprising: providing a monoclonal antibody or antigen binding portionthereof which binds to prostate specific membrane antigen (PSMA) andcompetes for binding to PSMA with a monoclonal antibody selected fromthe group consisting of a monoclonal antibody produced by a hybridomawith an ATCC accession number HB-12101, a monoclonal antibody producedby a hybridoma with an ATCC accession number HB-12127, and a monoclonalantibody produced by a hybridoma with an ATCC accession number HB-12126,wherein the antibody is bound to a cytotoxic drug of plant origin; andadministering the antibody or antigen binding portion thereof to asubject under conditions effective to treat prostate cancer.
 14. Themethod according to claim 1, wherein the monoclonal antibody or antigenbinding portion thereof competes for binding to PSMA with the monoclonalantibody produced by a hybridoma with an ATCC accession number HB-12126.15. The method according to claim 1, 11, 12 or 13, wherein themonoclonal antibody or antigen binding portion thereof is internalizedwith the PSMA.
 16. The method according to claim 1, 11, 12 or 13,wherein the antigen binding portion is selected from the groupconsisting of a Fab fragment, a F(ab′)2 fragment, and a Fv fragment. 17.The method according to claim 1, wherein the cytotoxic drug is selectedfrom the group consisting of a therapeutic drug, a compound emittingradiation, a molecule of plant, fungal, or bacterial origin, abiological protein, and a mixture thereof.
 18. The method according toclaim 1, wherein the cytotoxic drug is a compound emitting radiation.19. The method according to claim 18, wherein the compound emittingradiation is an alpha-emitter.
 20. The method according to claim 19,wherein the alpha-emitter is selected from the group consisting of²¹²Bi, ²¹³Bi, and ²¹¹At.
 21. The method according to claim 18, whereinthe compound emitting radiation is a beta-emitter.
 22. The methodaccording to claim 21, wherein the beta-emitter is ¹⁸⁶Re.
 23. The methodaccording to claim 21, wherein the beta-emitter is ⁹⁰Y.
 24. The methodaccording to claim 18, wherein the compound emitting radiation is agamma-emitter.
 25. The method according to claim 24, wherein thegamma-emitter is ¹³¹I.
 26. The method according to claim 1, wherein thecytotoxic drug is a molecule of bacterial origin.
 27. The methodaccording to claim 1, wherein the cytotoxic drug is a molecule of plantorigin.
 28. The method according to claim 1, wherein the cytotoxic drugis a biological protein.
 29. The method according to claim 1, whereinthe monoclonal antibody or antigen binding portion thereof furthercomprises a label.
 30. The method according to claim 1, wherein thelabel is selected from the group consisting of a biologically-activeenzyme label, and a radiolabel.
 31. The method according to claim 30,wherein the label is a radiolabel selected from the group consisting of¹¹¹In, ^(99m)Tc, ³²P, ¹²⁵I, ¹³¹I, ¹⁴C, ³H and ¹⁸⁸Rh.
 32. The methodaccording to claim 1, 11, 12 or 13, wherein the monoclonal antibody orantigen binding portion thereof administered to the subject is in acomposition further comprising a pharmaceutically acceptable carrier,excipient, or stabilizer.
 33. The method according to claim 1, 11, 12 or13 wherein the monoclonal antibody or antigen binding portion thereof isadministered in conjunction with a second therapeutic modality.
 34. Themethod according to claim 33, wherein the second therapeutic modality isselected from the group consisting of surgery, radiation, chemotherapy,immunotherapy and hormone replacement.
 35. The method according to claim34, wherein the hormone replacement comprises treatment with estrogen oran anti-androgen agent.
 36. The method according to claim 35, whereinthe anti-androgen agent is an agent which blocks or inhibits the effectsof testosterone.
 37. The method according to claim 12, wherein theprostate cancer is metastatic prostate cancer.
 38. The method accordingto claim 37, wherein the metastatic prostate cancer involves a bonemarrow or a lymph node metastasis.
 39. The method according to claim 12,wherein the administering is carried out parenterally.
 40. The methodaccording to claim 12, wherein the administering is carried outintravenously.
 41. The method according to claim 12, wherein theadministering is carried out by intracavitary instillation.
 42. Themethod according to claim 12, wherein the monoclonal antibody or antigenbinding portion thereof is administered following a prostatectomy. 43.The method according to claim 12, wherein the monoclonal antibody orantigen binding portion binds live cells.
 44. The method according toclaim 12, wherein the monoclonal antibody provided is produced by ahybridoma with an ATCC accession number HB-12101.
 45. The methodaccording to claim 12, wherein the monoclonal antibody or antigenbinding portion thereof competes for binding to PSMA with the monoclonalantibody produced by a hybridoma with an ATCC accession number HB-12126.46. The method according to claim 1, 10, 11, 12, or 13, wherein themethod of treating prostate cancer is a method that prevents theprogression of prostate cancer or delays the progression of prostatecancer in the subject.
 47. The method according to claim 1, wherein themonoclonal antibody provided is produced by a hybridoma with an ATCCaccession number HB-12127.
 48. The method according to claim 1, whereinthe monoclonal antibody provided is produced by a hybridoma with an ATCCaccession number HB-12126.
 49. The method according to claim 12, whereinthe monoclonal antibody provided is produced by a hybridoma with an ATCCaccession number HB-12127.
 50. The method according to claim 12, whereinthe monoclonal antibody provided is produced by a hybridoma with an ATCCaccession number HB-12126.